Pharrnacol.ogica/ Research Communications, Vol. 9, No. 9, 1977
847
BUPIVACAINE: MORPHOLOGICAL EFFECTS ON SPINAL CORDS OF CATS AND DURATIONS OF SPINAL ANESTHESIA IN SHEEP + H. Jack Adams, Ph.D.*, Angeline R. Mastri, M.D. and Dan Doherty, Jr.* *Research Department, Astra Pharmaceutical Products, Inc. Framingham, MA 01701 +Department of Neurology, University of Minnesota Minneapolis, ~ 55455
R~eived~fina/form 17 ~to~r1977
Bupivacaine HCI,
0.25,
~.5 and 1.0%, was administered
to cats and the duration of block recorded. gross and microscopi~ vealed morphological polyethylene
Subsequent
examination of the spinal cords rechanges due to the presence of the
catheter in the epidural space.
evidence of drug-induced morphological anesthesia with 0.25,
epidurally
There was no
changes.
Spinal
0.5 and 1.0% bupivacaine HCl was
performed in sheep, and durations of sensory analgesia and motor block were recorded.
Despite certain limitations,
inherent in all animal models,
the sheep is an excellent
model for the study of spinal anesthetic agents. Introduction The purposes of the studies reported here were to examine the morphological
effects,
if any, of bupivacaine on the
neuraxis of the cat after epidural administration
and to
PharrnacologicaPResearchComrnunications, Vol. 9, No. 9, 1977
848 evaluate
further the sheep as a m o d e l for the testing of
spinal anesthetic
agents.
Methods
Epidural
anesthesia
in cats and m i c r o s c o p i c
e x a m i n a t i o n ' of
the spinal cords. The m e t h o d
for surgical
administration
implantation
of local anesthetic
solutions
space of the cat has been d e s c r i b e d (1969).
In brief,
implanted distal
a polyethylene
end lay in the epidural
into the epidural
in detail by Duce et al.
catheter
into the 6th lumbar v e r t e b r a
filled with sterile s a l i n e end.
of a c a t h e t e r to p e r m i t
space.
(P.E. #20) was
so that 1.5 cm of the The catheter was
and h e a t - s e a l e d
at the p r o x i m a l
Injections were m a d e by cutting off the sealed tip and
attaching
a h y p o d e r m i c needle and syringe c o n t a i n i n g
local a n e s t h e t i c
solution or vehicle.
a dose v o l u m e of 1.5 ~i,
the
All animals received
injected at the rate of 0.5 m l / 2 0
The catheter was then flushed with s u f f i c i e n t to clear it of local a n e s t h e t i c
solution,
sterile saline
and the tip was
resealed.
The animal was observed
anesthesia
such as loss of weight support and block of the
flexor reflex effects.
sec.
for signs of onset of
in the hind limbs and for overt systemic
A n i m a l s were observed at 15 to 30 m i n u t e intervals
until complete r e - o v e r y from the epidural block. All animals,
i n c l u d i n g the saline controls,
were s a c r i f i c e d
by means of an O v e r d o s e of I.V. p e n t o b a r b i t a l
24 hours after
the epidural
c o l u m n was
dissected
injection.
The entire v e r t e b r a l
free as r a p i d l y as possible,
and the dorsal portions
Pharmacological Research Communications, Vol. 9, No. 9, 1977
849
of the vertebrae were r e m o v e d to permit rapid access of the fixing solution to the cord.
Care was taken,
however,
to
leave the 6th lumbar vertebra and the implanted catheter intact.
The columns were suspended in 10% buffered formalin
t h a t had been chilled to minimize autolysis.
All specimens
were identified by animal number only.
The preserved cords were examined grossly both externally and in multiple cross-sections. and lower thoracic segments)
The caudal one-third
(lumbosacral
of the spinal cord was serially
sectioned at 0.5 cm intervals and these sections examined microscopically.
The remainder of the spinal cord was
serially sectioned and representative sections from the cervical and thoracic cQrds removed for histologic examination. Sections Were stained routinely with hematoxylin and eosin, and selected sections were stained with Luxol fast blue-PAS and Bielschowsky silver stain.
Both the gross and the
microscopic examinations of the specimens w e r e d o n e
blind.
One male and seven female cats, weighing 3 to 4 kg, were used.
Each of the three concentrations of bupivacaine was
administered to two cats, and two received sterile isotonic saline.
Solutions were prepared by dissolving bupivacaine HCl in distilled water and autoclaving to insure sterility. centrations are given in terms of base.
The pH of the
bupivacaine solutions ranged from 5.4 to 5.7. sterile isotonic saline was 7.
Con-
The pH of the
Pharmacologica#Re~earch Communications, Vol. 9, No..9, 1977
850
Spinal anesthesia in the sheep The methods for performing spinal anesthesia in sheep and monitoring the blocks have been described in detail 1975).
(Lebeaux,
Fifteen non-pregnant ewes, weighing 36 to 54 kg, %
were selected for the study.
No animal was used more than
once. The sheep were placed in specially-designed
stocks to facilitate
giving the spinal injection and kept ther e until complete
! regression of the block occurred.
An area on the back 4 to
5 inches wide and extending from about L1 to S4 w a s clipped, and the region of the lumbosacral ~junct~on was scrubbed and disinfected.
Injections were made through the lumbosacral
interspace by means of I 22' gauge, spinal needles.
3-inch clear hub disposable
When a free flow of spinal fluid was evident,
the syringe was attached and 2 ml of local anesthetic solution injected without barbotage. As soon as the injection wa~ completed, the animal was examined for signs of onset of sensory analgesia~ and motor %
block.
As i s
done in
clinical
studies,-we
clamp to assess Sensory analgesia. sufficient
pressure
sensory-analgesia, tissues.
to
elicita
response
in-~the absence
a well-deveioped
spread and regression-of
analgesia" on"the d o r s ~ : 0 f the
Care was~-~aken;to apply of
skin twitch
reflex,~ and this permits .the_ ... observers_ ~to
follow the dermatomal
forceps
an Allis
but not enough t6Zcause injury eo the
The sheep~possesses
(fly-flicker)
used
presence
or
~e
absence
animal. of
sensor~.
By mga~so of Allis
sensory.analgesia
in-the
Pharmacological Research Communications, Vol. 9, No. 9, 1977 anal region and in the hind limbs was determined.
851 The Allis
forceps was also used on an unblocked area such as the forelimbs in order to elicit flexion of the tarsal joint of the hind limbs; hind limbs.
this was done to assess motor block in the
These variabies were checked at intervals until
complete recovery had occurred.
Since there are often
marked differences between the durations of block obtained on the left and right sides, these were recorded separately. Bupivacaine solutions were prepared by dissolving the HCl salt in 5% glucose and autoclaving to insure sterility. Concentrations are as the salt, and the pH ranged from 6.8 to 7.1.
The vials were coded so that the personnel injecting
the animals and making the observations were unaware of which concentration each animal received until the animal had completely recovered from the block. Results
Epidural anesthesia i n cats and microscopic examination of th e spinal cords.
Mean durations of loss of weight support in the hind limbs were 43, 120 and 236 m i n u t e s
for the 0.25,
concentrations respectively.
The
0.5 and 1.0%
flexor reflex was not
blocked in either cat at 0.25%; mean durations of block of the flexor reflex were 42~and 152 minutes at 0.5 and 1.0%. Duce et al.
(1969), using lidocaine and tetracaine,
also
J
found that duration of block of the flexor reflex was shorter than duration of loss of weight s u p p o r t .
No blocks occurred
in the animals that received isotonic sal~ne,
and all the
Pharrnaco/ogicat Research Communications, Vol. 9, No. 9, 1977
852 animals
that r e c e i v e d b u p i v a c a i n e
of normal
sensory and m o t o r
Microscopic
examination
logical changes confined cord,
showed complete r e c o v e r y
functions
of cord sections r e v e a l e d m o r p h o -
in all the cords,
but these changes were
to the lumbar and lumbosacral
i.e.,
in the hind limbs.
divisions
the site of the indwelling catheter.
of the Mast
commonly noted were c o m p r e s s i o n of the cord by the c a t h e t e r and focal
inflammation
dose-response: received
changes
and focal degeneration.
There is no
bn the cords of the animals that
1.0% b u p i v a c a i n e were not m o r e severe than in those
that received
0.25 or 0.5%.
In fact, m o r p h o l o g i c a l
in the cords of the two cats that received
changes
sterile isotonic
saline were as severe as in any of the other treatment groups.
Spinal a n e s t h e s i a in s h e e p The data are summarized
in Table I.
Sensory analgesia
m o t o r block show a clear d o s e - d u r a t i o n
relationship.
of m o t o r block was h i g h e r at 0.5% b u p i v a c a i n e however,
our e x p e r i e n c e
able variability.
is that this p a r a m e t e r
and Frequency
than at 1.0%; shows c o n s i d e r -
One of the five a n i m a l s at 0.25 and at
0.5% and 3/5 at 1.0% e x h i b i t e d an i n a b i l i t y
to support
t h e m s e l v e s on their hind limbs for several hours after c o m p l e t e r e g r e s s i o n of the block. were c o m p l e t e l y r e c o v e r e d
All animals,
the following day.
however,
_
.
1.0%
.
.
.
.
.
.
.
.
-
263
0.5%
.....
334
204
+ 78
+ 30
(80%)
99 + 13
Hind Limbs ii
No
(50%)
179 + ill
118 + 36
Block
Mean + S.D.
I
Motor Block
Frequency of block, if less than i00%, is given in parenthesis.
Durations are in minutes:
357 + 105
+ 71
150 + 57
Anal Region
Sensory Analgesia
0.25%
Concentration
Durations
+ 38
336 + 92
237
108 + 45
Regression
Complete Dermatonai o
Durations of Sensory Analgesia and Motor Block After Subarachnoid Administration of Bupivacaine In Sheep
Bupivacaine
Table I:
j
&O
D
Pharrnacologica/. Research Communications;. Vol.. 9,, No.. 9, 1977
854 Discussion
Epidural anesthesia in cats and microscopic examination of the spinal cords The presence of inflammatory and degenerative changes in the spinal cords of the control animals,
the observation that
these changes were restricted to the area of the indwelling cathether, and the absence of any dose-response relationship points to the catheter as the causative agent.
Drug-induced
morphological changes tend to be "patchy" and diffuse, and the severity of the changes increases as the concentration of drug is increased 1944).
(Adams et al., 1975; CoTui et al.,
We conclude, therefore,
that the morphological
changes in the spinal cord are due to the catheter's impinging on the cord and not to the local anesthetic agent.
We plan
in future studies of this type to modify the implantation of the catheter so that its tip opens into the epidural space but the catheter itself does not lie in the space. Spinal anesthesia in sheep Lebeaux
(1975) described the techniques for giving epidural
and subarachnoid injections in sheep and recommended this animal model for screening new local anesthetic agents and testing of agents "contemplated for injection into these spaces in human~."
Testing of bupivacaine at three concen-
trations in our study resulted in a dose-duration relationship, indicating this is indeed a good pharmacological model. Furthermore, the techniques used for evaluating sensory analgesia,
i.e., pinching with an Allis clamp, are similar
Pharmacological Research Communications, VoL 9, No. 9, 1977 to those used in clinical studies 1976).
The obvious difference
(Bridenbaugh
855 etal.,
is that in the case of the
animal model the subject and the observer cannot communicate with each other verbally.
Consequently,
the observer applies
noxious stimuli and infers from the presence or absence of withdrawal
and protective responses whether or not sensory
analgesia and motor block are present. limitations,
In spite of such
the observer can assess sensory analgesia and
motor block independently with a reasonable degree of certainty. For example,
at 0.25% bupivacaine
there was sensory analgesia
in the hind limbs of 4/5 animals but motor block in none, and motor block at 0.5 and 1.0% was consistently
shorter in
duration than sensory analgesia in the hind limbs. observations
These
are in good agreement with those made in clinical
studies and further emphasize
the validity of this animal
model for. evaluating agents used to produce central neural blocks.
References Adams, H.J., A.R. Mastri, A.W. Eicholzer and G. Kilpatrick. Anesth. Analg. 53:904-908 (1974) Bridenbaugh, Anesthesiol.
P.O., R.I. Balfour, 45:560-564 (1976)
L.D. Bridenbaugh
and D.F. Lysons.
CoTui, F.W., A.L. Preiss, I. Barcham and M.I. Nevin. J. Pharmacol. Exptl, T h e r ~ 81:209-217 (1944) Duce, B.R., K. Zelechowski, G. Camougis and E.R. Smith. Brit. J. Anaesth. 41:579-587 (1969) Lebeaux, M.1. Lab. Animal Sci.
25:629-633
(1975)
847
BUPIVACAINE: MORPHOLOGICAL EFFECTS ON SPINAL CORDS OF CATS AND DURATIONS OF SPINAL ANESTHESIA IN SHEEP + H. Jack Adams, Ph.D.*, Angeline R. Mastri, M.D. and Dan Doherty, Jr.* *Research Department, Astra Pharmaceutical Products, Inc. Framingham, MA 01701 +Department of Neurology, University of Minnesota Minneapolis, ~ 55455
R~eived~fina/form 17 ~to~r1977
Bupivacaine HCI,
0.25,
~.5 and 1.0%, was administered
to cats and the duration of block recorded. gross and microscopi~ vealed morphological polyethylene
Subsequent
examination of the spinal cords rechanges due to the presence of the
catheter in the epidural space.
evidence of drug-induced morphological anesthesia with 0.25,
epidurally
There was no
changes.
Spinal
0.5 and 1.0% bupivacaine HCl was
performed in sheep, and durations of sensory analgesia and motor block were recorded.
Despite certain limitations,
inherent in all animal models,
the sheep is an excellent
model for the study of spinal anesthetic agents. Introduction The purposes of the studies reported here were to examine the morphological
effects,
if any, of bupivacaine on the
neuraxis of the cat after epidural administration
and to
PharrnacologicaPResearchComrnunications, Vol. 9, No. 9, 1977
848 evaluate
further the sheep as a m o d e l for the testing of
spinal anesthetic
agents.
Methods
Epidural
anesthesia
in cats and m i c r o s c o p i c
e x a m i n a t i o n ' of
the spinal cords. The m e t h o d
for surgical
administration
implantation
of local anesthetic
solutions
space of the cat has been d e s c r i b e d (1969).
In brief,
implanted distal
a polyethylene
end lay in the epidural
into the epidural
in detail by Duce et al.
catheter
into the 6th lumbar v e r t e b r a
filled with sterile s a l i n e end.
of a c a t h e t e r to p e r m i t
space.
(P.E. #20) was
so that 1.5 cm of the The catheter was
and h e a t - s e a l e d
at the p r o x i m a l
Injections were m a d e by cutting off the sealed tip and
attaching
a h y p o d e r m i c needle and syringe c o n t a i n i n g
local a n e s t h e t i c
solution or vehicle.
a dose v o l u m e of 1.5 ~i,
the
All animals received
injected at the rate of 0.5 m l / 2 0
The catheter was then flushed with s u f f i c i e n t to clear it of local a n e s t h e t i c
solution,
sterile saline
and the tip was
resealed.
The animal was observed
anesthesia
such as loss of weight support and block of the
flexor reflex effects.
sec.
for signs of onset of
in the hind limbs and for overt systemic
A n i m a l s were observed at 15 to 30 m i n u t e intervals
until complete r e - o v e r y from the epidural block. All animals,
i n c l u d i n g the saline controls,
were s a c r i f i c e d
by means of an O v e r d o s e of I.V. p e n t o b a r b i t a l
24 hours after
the epidural
c o l u m n was
dissected
injection.
The entire v e r t e b r a l
free as r a p i d l y as possible,
and the dorsal portions
Pharmacological Research Communications, Vol. 9, No. 9, 1977
849
of the vertebrae were r e m o v e d to permit rapid access of the fixing solution to the cord.
Care was taken,
however,
to
leave the 6th lumbar vertebra and the implanted catheter intact.
The columns were suspended in 10% buffered formalin
t h a t had been chilled to minimize autolysis.
All specimens
were identified by animal number only.
The preserved cords were examined grossly both externally and in multiple cross-sections. and lower thoracic segments)
The caudal one-third
(lumbosacral
of the spinal cord was serially
sectioned at 0.5 cm intervals and these sections examined microscopically.
The remainder of the spinal cord was
serially sectioned and representative sections from the cervical and thoracic cQrds removed for histologic examination. Sections Were stained routinely with hematoxylin and eosin, and selected sections were stained with Luxol fast blue-PAS and Bielschowsky silver stain.
Both the gross and the
microscopic examinations of the specimens w e r e d o n e
blind.
One male and seven female cats, weighing 3 to 4 kg, were used.
Each of the three concentrations of bupivacaine was
administered to two cats, and two received sterile isotonic saline.
Solutions were prepared by dissolving bupivacaine HCl in distilled water and autoclaving to insure sterility. centrations are given in terms of base.
The pH of the
bupivacaine solutions ranged from 5.4 to 5.7. sterile isotonic saline was 7.
Con-
The pH of the
Pharmacologica#Re~earch Communications, Vol. 9, No..9, 1977
850
Spinal anesthesia in the sheep The methods for performing spinal anesthesia in sheep and monitoring the blocks have been described in detail 1975).
(Lebeaux,
Fifteen non-pregnant ewes, weighing 36 to 54 kg, %
were selected for the study.
No animal was used more than
once. The sheep were placed in specially-designed
stocks to facilitate
giving the spinal injection and kept ther e until complete
! regression of the block occurred.
An area on the back 4 to
5 inches wide and extending from about L1 to S4 w a s clipped, and the region of the lumbosacral ~junct~on was scrubbed and disinfected.
Injections were made through the lumbosacral
interspace by means of I 22' gauge, spinal needles.
3-inch clear hub disposable
When a free flow of spinal fluid was evident,
the syringe was attached and 2 ml of local anesthetic solution injected without barbotage. As soon as the injection wa~ completed, the animal was examined for signs of onset of sensory analgesia~ and motor %
block.
As i s
done in
clinical
studies,-we
clamp to assess Sensory analgesia. sufficient
pressure
sensory-analgesia, tissues.
to
elicita
response
in-~the absence
a well-deveioped
spread and regression-of
analgesia" on"the d o r s ~ : 0 f the
Care was~-~aken;to apply of
skin twitch
reflex,~ and this permits .the_ ... observers_ ~to
follow the dermatomal
forceps
an Allis
but not enough t6Zcause injury eo the
The sheep~possesses
(fly-flicker)
used
presence
or
~e
absence
animal. of
sensor~.
By mga~so of Allis
sensory.analgesia
in-the
Pharmacological Research Communications, Vol. 9, No. 9, 1977 anal region and in the hind limbs was determined.
851 The Allis
forceps was also used on an unblocked area such as the forelimbs in order to elicit flexion of the tarsal joint of the hind limbs; hind limbs.
this was done to assess motor block in the
These variabies were checked at intervals until
complete recovery had occurred.
Since there are often
marked differences between the durations of block obtained on the left and right sides, these were recorded separately. Bupivacaine solutions were prepared by dissolving the HCl salt in 5% glucose and autoclaving to insure sterility. Concentrations are as the salt, and the pH ranged from 6.8 to 7.1.
The vials were coded so that the personnel injecting
the animals and making the observations were unaware of which concentration each animal received until the animal had completely recovered from the block. Results
Epidural anesthesia i n cats and microscopic examination of th e spinal cords.
Mean durations of loss of weight support in the hind limbs were 43, 120 and 236 m i n u t e s
for the 0.25,
concentrations respectively.
The
0.5 and 1.0%
flexor reflex was not
blocked in either cat at 0.25%; mean durations of block of the flexor reflex were 42~and 152 minutes at 0.5 and 1.0%. Duce et al.
(1969), using lidocaine and tetracaine,
also
J
found that duration of block of the flexor reflex was shorter than duration of loss of weight s u p p o r t .
No blocks occurred
in the animals that received isotonic sal~ne,
and all the
Pharrnaco/ogicat Research Communications, Vol. 9, No. 9, 1977
852 animals
that r e c e i v e d b u p i v a c a i n e
of normal
sensory and m o t o r
Microscopic
examination
logical changes confined cord,
showed complete r e c o v e r y
functions
of cord sections r e v e a l e d m o r p h o -
in all the cords,
but these changes were
to the lumbar and lumbosacral
i.e.,
in the hind limbs.
divisions
the site of the indwelling catheter.
of the Mast
commonly noted were c o m p r e s s i o n of the cord by the c a t h e t e r and focal
inflammation
dose-response: received
changes
and focal degeneration.
There is no
bn the cords of the animals that
1.0% b u p i v a c a i n e were not m o r e severe than in those
that received
0.25 or 0.5%.
In fact, m o r p h o l o g i c a l
in the cords of the two cats that received
changes
sterile isotonic
saline were as severe as in any of the other treatment groups.
Spinal a n e s t h e s i a in s h e e p The data are summarized
in Table I.
Sensory analgesia
m o t o r block show a clear d o s e - d u r a t i o n
relationship.
of m o t o r block was h i g h e r at 0.5% b u p i v a c a i n e however,
our e x p e r i e n c e
able variability.
is that this p a r a m e t e r
and Frequency
than at 1.0%; shows c o n s i d e r -
One of the five a n i m a l s at 0.25 and at
0.5% and 3/5 at 1.0% e x h i b i t e d an i n a b i l i t y
to support
t h e m s e l v e s on their hind limbs for several hours after c o m p l e t e r e g r e s s i o n of the block. were c o m p l e t e l y r e c o v e r e d
All animals,
the following day.
however,
_
.
1.0%
.
.
.
.
.
.
.
.
-
263
0.5%
.....
334
204
+ 78
+ 30
(80%)
99 + 13
Hind Limbs ii
No
(50%)
179 + ill
118 + 36
Block
Mean + S.D.
I
Motor Block
Frequency of block, if less than i00%, is given in parenthesis.
Durations are in minutes:
357 + 105
+ 71
150 + 57
Anal Region
Sensory Analgesia
0.25%
Concentration
Durations
+ 38
336 + 92
237
108 + 45
Regression
Complete Dermatonai o
Durations of Sensory Analgesia and Motor Block After Subarachnoid Administration of Bupivacaine In Sheep
Bupivacaine
Table I:
j
&O
D
Pharrnacologica/. Research Communications;. Vol.. 9,, No.. 9, 1977
854 Discussion
Epidural anesthesia in cats and microscopic examination of the spinal cords The presence of inflammatory and degenerative changes in the spinal cords of the control animals,
the observation that
these changes were restricted to the area of the indwelling cathether, and the absence of any dose-response relationship points to the catheter as the causative agent.
Drug-induced
morphological changes tend to be "patchy" and diffuse, and the severity of the changes increases as the concentration of drug is increased 1944).
(Adams et al., 1975; CoTui et al.,
We conclude, therefore,
that the morphological
changes in the spinal cord are due to the catheter's impinging on the cord and not to the local anesthetic agent.
We plan
in future studies of this type to modify the implantation of the catheter so that its tip opens into the epidural space but the catheter itself does not lie in the space. Spinal anesthesia in sheep Lebeaux
(1975) described the techniques for giving epidural
and subarachnoid injections in sheep and recommended this animal model for screening new local anesthetic agents and testing of agents "contemplated for injection into these spaces in human~."
Testing of bupivacaine at three concen-
trations in our study resulted in a dose-duration relationship, indicating this is indeed a good pharmacological model. Furthermore, the techniques used for evaluating sensory analgesia,
i.e., pinching with an Allis clamp, are similar
Pharmacological Research Communications, VoL 9, No. 9, 1977 to those used in clinical studies 1976).
The obvious difference
(Bridenbaugh
855 etal.,
is that in the case of the
animal model the subject and the observer cannot communicate with each other verbally.
Consequently,
the observer applies
noxious stimuli and infers from the presence or absence of withdrawal
and protective responses whether or not sensory
analgesia and motor block are present. limitations,
In spite of such
the observer can assess sensory analgesia and
motor block independently with a reasonable degree of certainty. For example,
at 0.25% bupivacaine
there was sensory analgesia
in the hind limbs of 4/5 animals but motor block in none, and motor block at 0.5 and 1.0% was consistently
shorter in
duration than sensory analgesia in the hind limbs. observations
These
are in good agreement with those made in clinical
studies and further emphasize
the validity of this animal
model for. evaluating agents used to produce central neural blocks.
References Adams, H.J., A.R. Mastri, A.W. Eicholzer and G. Kilpatrick. Anesth. Analg. 53:904-908 (1974) Bridenbaugh, Anesthesiol.
P.O., R.I. Balfour, 45:560-564 (1976)
L.D. Bridenbaugh
and D.F. Lysons.
CoTui, F.W., A.L. Preiss, I. Barcham and M.I. Nevin. J. Pharmacol. Exptl, T h e r ~ 81:209-217 (1944) Duce, B.R., K. Zelechowski, G. Camougis and E.R. Smith. Brit. J. Anaesth. 41:579-587 (1969) Lebeaux, M.1. Lab. Animal Sci.
25:629-633
(1975)
